Topical aminolevulinic acid-photodynamic therapy for the treatment of acne vulgaris

ABSTRACT

Light treatments of sebaceous gland disorders with 5-aminolevulinic acid and photodynamic therapy are disclosed. A preferred treatment includes topical application of 5-aminolevulinic acid to the skin followed by light exposures with repeated treatment at various intervals. At low doses of ALA and photodynamic therapy (PDT) in single or multiple treatments, improvement in the sebaceous gland disorder, e.g., acne, provides the discovery that diminishment in sebum secretion and the eradication of bacteria occurs. At high doses of ALA and a single high energy PDT treatment, permanent changes to the sebaceous gland and sebum secretion have been discovered.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/535,937, filed on Aug. 5, 2009 and entitled “Topical AminolevulinicAcid-Photodynamic Therapy for the Treatment of Acne Vulgaris,” which isa continuation of U.S. patent application Ser. No. 10/970,922, filed onOct. 20, 2004 and entitled “Topical Aminolevulinic Acid-PhotodynamicTherapy for the Treatment of Acne Vulgaris,” now abandoned, which is adivisional of U.S. patent application Ser. No. 09/929,384, filed on Aug.14, 2001 and entitled “Topical Aminolevulinic Acid-Photodynamic TherapyFor The Treatment Of Acne Vulgaris,” now issued as U.S. Pat. No.6,897,238, which claims priority to U.S. Provisional Patent ApplicationNo. 60/225,691, filed Aug. 16, 2000 and entitled “Topical AminolevulinicAcid-Photodynamic Therapy For The Treatment Of Acne Vulgaris,” all ofwhich are incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Skin disorders, such as acne, can be irritating and embarrassing. Themajor disease of skin associated with sebaceous follicles, is acnevulgaris. This is also the most common reason for visiting adermatologist in the United States. There are many treatments, but nocures for acne. These include antibiotics (which inhibit growth of p.acnes bacteria which play a role in acne), retinoids such as Accutane®(isotetinoin, which reduces sebaceous gland output of sebum), andantimicrobials such as benzoyl peroxide. Acne lesions result from therupture of a sebaceous follicle, followed by inflammation and pus (a“whitehead”), or by accumulation of plugged material in the sebaceousfollicle (a “blackhead”). This pathophysiology has two majorrequirements: (1) plugging of the upper portion of the follicle, and (2)an increase in sebum production. The upper portion of the follicle,i.e., the “pore” into which sebum is secreted and which is directly incommunication with the skin surface, is called the infundibulum. A plugforms in the infundibulum from cells, sebum, bacteria, and other debris.The sebaceous gland continues to produce sebum (an oily fluid),stretching the infundibulum until either it or some lower portion of thefollicles ruptures.

Generally, only a minority of sebaceous hair follicles on the face andupper back develop acne lesions. Therefore, it is likely that somestructural differentiation predisposes a fraction of the follicles todevelop acne. In most males, acne is worst in the teenage years and thensubsides, suggesting that a subpopulation of follicles may be presentwhich ultimately self-destruct. In women, teenage acne is often followedby menstrual acne flares well into adulthood. Since both plugging of theinfundibulum and high sebaceous gland activity are necessary for an acnelesion to develop, it is likely that two of the predisposing factors forthe follicles which become infected are (1) an infundibulum shape whichis easily plugged, and/or (2) a hyperactive sebaceous gland.

Unlike medical dermatology, most laser dermatology treatments areactually “cures”—producing a permanent anatomic, microsurgical effect onthe skin. This includes skin resurfacing, portwine stain treatment,tattoo and pigmented lesion removal, and hair removal. Selectivephotothermolysis or controlled skin ablation with lasers or otherextremely intense light sources, might therefore be capable of curingskin disorders, such as acne, if appropriately targeted to the primarysite(s) of pathophysiology.

Therefore a need exists which circumvents and provides a solution to theabove-described shortcomings of the presently known treatments.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that5-aminolevulinic acid (ALA) described infra, in combination with anenergy source, e.g., photo (light) therapy, can be used to modulate,e.g., treat, sebaceous gland disorders, e.g., eliminate, inhibit, orprevent occurrence or reoccurrence of the skin disorder.Topically-applied ALA is taken up by epithelial cells and metabolizedvia the porphyrin pathway to protoporphyrin IX (PpIX), the precursor ofheme. PpIX is a photosensitizer that accumulates not only in theepidermal cells, but also the pilosebaceous units. When intense light,e.g., visible light, red light, light with a wavelength range of betweenabout 320 and 700 nm, is delivered to the ALA-treated skin, PpIX isexcited into a triplet state, which reacts with oxygen to producesinglet oxygen, causing membrane damage and cell destruction. TopicalALA may directly enter hair follicles, where sebaceous glands activelysynthesize and retain PpIX.

The present treatment protocol is efficient, is topical, and providesrelief of the sebaceous gland disorder for at least 20 weeks. Moreover,the present invention provides optimized conditions for treatment ofskin, such that the therapeutic treatment is non-irritating, longlasting (greater than 20 weeks) and can be accomplished in one or moreapplications. A preferred example of such a sebaceous gland disorder isacne.

The present invention pertains to methods for treating skin disordersassociated with sebaceous follicles by topically applying5-aminolevulinic acid (ALA) to a section of skin afflicted with asebaceous gland disorder, wherein the ALA is converted into PpIX whichis then activated by energy that penetrates outer layers of epidermis. Asufficient amount of the ALA infiltrates the afflicted section of skin,is converted into PpIX, and is exposed to sufficient energy to cause thePpIX to become photodynamically activated, thereby treating thesebaceous gland disorder. In one embodiment, the sebaceous glanddisorder is acne. Suitable energy sources for photodynamic treatmentinclude flash lamp based sources and lasers, such as Nd:YAG,Alexandrite, flash lamp-pumped dyes and diodes. Alternatively, theenergy source can also be a continuous wave energy source. In preferredembodiments, the ALA is dissolved in an aqueous/alcoholic solution inconcentrations between about 10% and 20% by weight.

The present invention also pertains to methods for modifying the openingto the infundibulum by topically applying ALA to the opening to theinfundibulum, wherein the ALA is converted into PpIX, that is thenphotodynamically activated by energy which penetrates outer layers ofepidermis. A sufficient amount of the ALA infiltrates spaces about theinfundibulum and the infundibulum is exposed to sufficient energy tocause the converted ALA to become photodynamically activated, therebymodifying the opening to the infundibulum. In one embodiment, theopening to the infundibulum is increased. In still another embodiment,the opening to the infundibulum is altered such that pore pluggage willnot occur, e.g., the infundibulum is reshaped such that excess sebum,oils, dirt and bacteria will not cause pore pluggage to occur, resultingin a black head (comedon) or white head (milium).

The present invention also pertains to methods for suppressing, e.g.,decreasing, the oil/lipid output production of the sebaceous gland.Application of ALA to the pilosebaceous unit, e.g., the sebaceous gland,followed by photodynamic stimulation of the resultant PpIX by an energysource can cause selective permanent physical alteration to thesebaceous gland and/or follicle such that surrounding tissue remainsunaffected. The physical alteration to the sebaceous gland and/orfollicle results in diminished production of sebum and the size of thesebaceous gland is decreased.

The present invention further pertains to methods for modifying thepilosebaceous unit by topically applying ALA to the pilosebaceous unit,wherein the resultant PpIX is photodynamically activated by energy whichpenetrates into the dermis and into the outer layers of epidermis. Asufficient amount of ALA infiltrates the pilosebaceous unit and thepilosebaceous unit is exposed to sufficient energy to cause theincreased levels of PpIX to become photodynamically activated, therebymodifying the pilosebaceous unit. In one embodiment, the pilosebaceousunit is treated such that sebum production is diminished. A decrease inpore pluggage can occur, as a result of the diminishment of sebumproduction. In one preferred embodiment, treatment of the pilosebaceousunit by the present invention results in elimination of pore pluggage,e.g., the pilosebaceous unit is treated such that excess sebum, oils,dirt and bacteria will not cause pore pluggage to occur, resulting in ablack or white head.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, advantages and features of the present invention will bereadily appreciated as the same becomes better understood by referenceto the following detailed description when considered in connection withthe accompanying drawings, in which like reference numerals designatelike parts throughout the figures thereof and wherein:

FIG. 1 shows transient acneiform eruption caused by a single PDTtreatment, a) Baseline, b) 1 week post treatment;

FIG. 2 graphically represents the mean improvement (±SEM) by treatmentsites, treatment groups, and follow-up visits, a) Reduction ininflammatory acne score, b) Global clinical-improvement grading, c)Reduction in autofluorescence of follicles, related to P. acnes, and d)Reduction in sebum excretion rate;

FIG. 3 demonstrates inflammatory acne improved by a single PDTtreatment, a) Baseline, b) 10 weeks post PDT, c) Acne starts to resume20 weeks after PDT, Long-term remission of acne after multiple PDTtreatments. d) Baseline, e) 2 weeks post PDT (an irritation reaction toSebutape is seen on the right side in this subject), f) 20 weeks postPDT;

FIG. 4 shows the fluorescence of porphyrin from bacteria in follicles(red dots) decrease after a single ALA-PDT, Photography was taken asdescribed at baseline (a), week 2 (b), week 10 (c), and week 20 (d)post-PDT;

FIG. 5 demonstrates that sebum excretion is suppressed by a singleALA-PDT application, then gradually recovers, at baseline (a), week 2(b), week 10 (c), week 20 (d) post-PDT;

FIG. 6 demonstrates that Sebum excretion remains suppressed aftermultiple ALA-PDT treatments, for at least 20 weeks, (a) (b) (c) (d) asin FIG. 8;

FIG. 7 depicts retiform degeneration of sebocytes (a) and intense mixedneutrophil-predominant infiltrate (b), the biopsies were takenimmediately after a single ALA-PDT treatment. Scale bars, 100 μm;

FIG. 8 shows that neutrophillic pustules are seen 3 days after ALA-PDT,intraepidermally, (a) and within pilosebaceous units (b), associatedwith the acneiform eruption caused by ALA-PDT. The sections were stainedwith hemotoxylin & eosin (a & b), Scale bars, 100 μm;

FIG. 9 depicts focal vacuolization of sebocytes and follicularkeratinocytes, this biopsy was taken at week 3 from singleALA-PDT-treated skin, note the mild perifollicular fibrosis, Scale bar,100 μm;

FIG. 10 is a graphical representation of long-term damaged pilosebaceousunit caused by ALA-PDT, twenty weeks after 4 ALA-PDT treatments, thereare atrophic or partially damaged sebaceous glands (a), a granulomatousreaction in completely destroyed sebaceous glands (b), obliterated hairfollicles (c), and perifollicular fibrosis (c). Scale bars, 100 μm;

FIG. 11 are Fluorescence Micrographs, a) Fluorescence microscopydemonstrates PpIX production is mainly located in sebaceous glands (S)and hair follicles (F), Fluorescence is greater in sebaceous glands,compared to that in hair follicles; and

FIG. 12 demonstrates that ALA-induced PpIX fluorescence is greater inacne lesions than surrounding tissue.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

5-Aminolevulinic acid, also known as 5-aminolaevulinic acid,delta-aminolevulinic acid, delta-aminolaevulinic acid ,or5-amino-4-oxopentanoic acid, is an intermediate in the pathway to theproduction of the photosensitizer, proptoporphyrin IX (PpIX). In thepresent invention, 5-Aminolevulinic acid can be used as a salt, such asthe hydrochloride salt. 5-Aminolevulinic acid can also be used in apharmacologically equivalent form, such as an amide or ester. Examplesof precursors and products of 5-aminolevulinic acid andpharmacologically equivalent forms of 5-aminolevulinic acid that can beused in the present invention are described in J. Kloek et al., Prodrugsof 5-Aminolevulinic Acid for Photodynamic Therapy, Photochemistry andPhotobiology, Vol. 64 No. 6, December 1996, pages 994-1000; WO 95/07077;Q. Peng et al., Build-Up of EsterifiedAminolevulinic-Acid-Derivative-Induced Porphyrin Fluorescence in NormalMouse Skin, Journal of Photochemistry and Photobiology B: Biology, Vol.34, No. 1, June 1996; and WO 94/06424. These references are incorporatedherein in their entirety. The term “ALA” refers to all of theabove-referenced compounds as described herein.

The present invention is based, at least in part, on the discovery thatALA leads to increased concentration of PpIX in epithelial cells, hairfollicles, the pilosebaceous unit, the infundibulum and/or sebaceousglands, and in combination with an energy source, photodynamic therapycan be used to treat sebaceous gland disorders, e.g., eliminate, remove,or prevent occurrence or reoccurrence of the sebaceous gland disorder.Examples of such sebaceous gland disorders include sebaceous glandhyperplasia, acne vulgaris and acne rosacea. A preferred example of sucha sebaceous gland disorder is acne.

The term “photodynamic”refers to the administration of aphotosensitizing agent to a subject, including administration of aprecursor of a photosensitizing agent such as ALA, and subsequentirradiation with energy, e.g., light, of the target cells or tissue ofthe subject. It is believed that ALA and hence, the photosensitizingagent preferentially accumulate in the target cells, because they are ofan infective origin, e.g., bacteria. It has now been surprisinglydiscovered that the administration of ALA, as a result of their morerapid proliferation, causes the target cells or tissue containrelatively greater concentrations of light sensitive porphyrins, e.g.,PpIX, and thus are more sensitive to light. Thus, the targeted tissue(hair follicles, infundibulum, sebaceous gland, pilosebaceous unit)containing sufficiently high concentrations of the photosensitizingagent, including the metabolites of ALA, selectively absorb greateramounts of energy and can be selectively localized and distinguishedfrom the adjacent cells or tissues. Photodynamic activation of thephotosensitizing agent destroys the cells/tissue with increasedconcentrations of the photosensitizing agent. In particularly preferredembodiments, bacteria present in the sebaceous gland are eradicated. Theeffect of the light is dependent upon the photosensitizer selectedwavelength or range of wavelengths, as well as the intensity andduration of administration of the energy, e.g., light.

In one aspect, the present invention is drawn to methods for treatingsebaceous gland disorders by topically applying ALA to a section of skinafflicted with a sebaceous gland disorder. The ALA is converted in PpIXvia the protoporphyrin pathway, and the resultant photosensitizer PpIXis energetically stimulated by an energy source. A sufficient amount ofALA infiltrates the skin and the section of skin is exposed to at leastone frequency band of energy so as to impart, to the converted ALA,sufficient energy to cause the resultant PpIX to become photodynamicallyactivated resulting in a physiological change, thereby treating thesebaceous gland disorder. In one embodiment, the sebaceous glanddisorder is acne. Suitable energy sources include a wide range of pulsedor continuous electromagnetic sources including, optical energy emittedby the sun, ultraviolet light generators, flash lamp based sources andlasers, such as Nd:YAG, Alexandrite, and flash lamp-pumped dye lasersand diode lasers. Alternatively, the energy source can be a continuouswave energy source, such as arc lamps, tungsten-halogen lamps andlight-emitting diodes.

In preferred embodiments, the energy source emits visible light,especially red visible light. Generally, the range of energy applied tothe skin surface ranges from 1 J/cm² to about 200 J/cm², preferably fromabout 25 J/cm² to about 200 J/cm², and most preferably about 100 J/cm².

In general, the wavelength range for therapeutic treatment is from about320 nm to about 700 nm, preferably from about 550 to about 700 nm, morepreferably from about 550 to about 600 nm.

For example, in one embodiment, the skin is treated with a low dose ofALA and low dose of energy to provide relief from acne. This can beconsidered a therapeutic treatment in which occasional multipletreatments are required to alleviate the sebaceous gland disorder, e.g.,acne. The procedure can be repeated daily, monthly, bimonthly, everythree months or as required to maintain the diminishment of thesebaceous gland disorder. A suitable treatment includes topicalapplication of about 0.1 to about 10 weight percent of ALA, preferablybetween about 0.1 to about 5 weight percent, most preferably betweenabout 0.1 to 1 weight percent of ALA followed by a low dosage of energy,e.g., a range of between about 1 J/cm² and about 20 J/cm², preferablybetween about 1 J/cm² and about 10 J/cm², and most preferably betweenabout 1 J/cm² and 5 J/cm², e.g., 1 J/cm². This therapeutic treatment isof great interest in that these lower levels of ALA are effect todestroy the bacteria associated with acne; the bacteria is verysensitive to the ALA-photodynamic therapy. This therapy offers theadvantage of utilizing low levels of ALA and energy, such that thepatient does not feel discomfort and that the skin becomeshyperpigmented. The individual can undergo treatments on a regular basisto prevent or alleviate the sebaceous gland disorder. In general, theenergy utilized has a wavelength range of between about 330 nm and about650 nm to about 700 nm.

In another embodiment, the skin is treated with a high dose of ALA and ahigh dose of energy to provide a permanent improvement in the sebaceousgland disorder, e.g., acne. This can be viewed as a permanenttherapeutic cure for the affliction in that the sebaceous gland isdiminished in size and microscarring occurs to and about the sebaceousgland, thereby decreasing or eliminating the secretion of sebum. It isbelieved that the microscarring from the ALA-PDT therapy fixes the sizeof the sebaceous gland so that it cannot expand to once again producelarge quantities, relatively, of sebum. The microscarring and thereduction of sebaceous gland size and sebum production has been 6 monthsafter a single treatment. A suitable permanent treatment includestopical application of about 10 to about 30 weight percent of ALA,preferably between about 10 and about 20 weight percent ALA, and mostpreferably about 20 weight percent ALA followed by a high dose ofenergy, e.g., a range of between about 50 J/cm² and about 200 J/cm²,preferably between about 100 J/cm² and about 150 J/cm², and mostpreferably between about 125 J/cm² and 175 J/cm². In this embodiment,the optimal wavelength range is between about 550 nm and about 650 nm.

Typically, ALA is administered topically as a solution. Theconcentration of the ALA can be in the range from about 0.1 to about 30percent by weight, preferably from about 0.1 to about 20 percent byweight, and most preferably from about 10 to about 20 percent by weight.The ALA can be formulated into various creams and emulsions that canpenetrate into the skin. A preferred solution is a combination ofalcohol, ethyl alcohol and water. Generally, ALA is applied topically inan appropriate carrier and permitted to permeate into the skin over aperiod of about 1 to about 12 hours, preferably from about 2 to about 5hours, and most preferably about 3 hours. As a general practice, thetreated area is covered with material, such as a plastic, which helps toslow evaporation of the solvent of the carrier system. The individual isthen subjected to photodynamic therapy to treat the sebaceous glanddisorder. In one embodiment, the individual is treated with a 20% ALA ina hydroalcoholic vehicle (Levulan, provided by DUSA Pharmaceuticals) for3 hours under occlusion with plastic film and 150 J/cm² of broad bandlight (550-700 nm).

The present invention also pertains to methods for modifying the openingto the infundibulum by topically applying ALA to the opening to theinfundibulum, wherein the ALA is converted into PpIX, and is treatedwith at least one frequency band of energy which penetrates outer layersof epidermis. A sufficient amount of ALA infiltrates spaces about theinfundibulum and the section of skin is exposed to at least onefrequency band of energy so as to impart to the converted ALA, PpIX,sufficient energy to cause the PpIX to become photodynamicallyactivated, thereby modifying the opening to the infundibulum. In oneembodiment, the opening to the infundibulum is altered such that porepluggage will not occur, e.g., the infundibulum is reshaped such thatexcess sebum, oils, dirt and bacteria will not cause pore pluggage tooccur, resulting in a blackhead (comedon) or white head (milium). In apreferred embodiment, the opening to the infundibulum is opened.

The present invention further pertains to methods for modifying thepilosebaceous unit by topically applying ALA to the pilosebaceous unit,wherein the resultant PpIX absorbs at least one frequency band of energywhich penetrates outer layers of epidermis. A sufficient amount of theALA infiltrates the pilosebaceous unit and the section of skin isexposed to at least one frequency band of energy so as to impart to theresulting increased concentration of PpIX, sufficient energy to causethe PpIX to become photodynamically activated, thereby modifying thepilosebaceous unit. In one embodiment, the pilosebaceous unit is treatedsuch that sebum production is diminished, thereby resulting in decreasedpore pluggage. In one preferred embodiment, treatment of thepilosebaceous unit by the present invention results in elimination ofpore pluggage, e.g., the pilosebaceous unit is treated such that excesssebum, oils, dirt and bacteria will not cause pore pluggage to occur,resulting in a black or white head.

In another aspect, the invention includes the combination of ALA withUVA and/or UVB absorbing substances. The combination can be appliedtopically and is useful in the treatment of sebaceous gland disorders,such as acne. Application of the ALA with the UVA and/or UVB absorbingsubstance followed by PDT causes the converted ALA to eradicate bacteriaassociated with acne. Generally, sunlight is the energy source for PDTstimulation and the total fluence of energy is between about 1 and about100 J/cm², preferably between about 1 and about 50 J/cm² and mostpreferably between about 10 and about 40 J/cm². Typically the ALAconcentration is in the range of between about 0.1 to about 10 percentby weight, preferably between about 0.1 and about 5 percent by weightand most preferably between about 0.1 and 1 percent by weight. Ingeneral the UVA and/or UVB filter substances are included in thecomposition in a range of between about 0.1 to about 30% by weight,preferably from about 0.1 to about 10% by weight and most preferablyfrom about 0.1 to about 5% by weight.

Suitable UVB filters include those which absorb energy between about 290nm and 320 nm, the so-called UVB range, and are generally derivatives of3-benzylidene camphor, 4-aminobenzoic acid, cinnamic acid, salicylicacid, benzophenone and 2-phenylbenzimidazole. Examples of oil solubleUVB filters include 3-benzylidene camphor derivatives, e.g.,3-(4-methylbenzylidene)camphor, 3-benzylidene-camphor; 4-aminobenzoicacid derivatives, e.g., 4-(dimethylamino)-benzoic-acid(2-ethylhexyl)ester, 4-(dimethylamino)benzoic-acid-amylester; esters ofcinnamic acid, e.g., 4-methoxycinnamic-acid-(2-ethylhexyl)ester,4-methoxycinnamic-acid-isopentylester; esters of salicylic acids, e.g.,salicylic acid(2-ethylhexyl)ester, salicylicacid(4-isopropylbenzyl)ester, salicylic acid-homomenthylester;derivatives of benzophenone, e.g., 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acids,e.g., 4-methoxybenzalmalonic-acid-di(2-ethylhexyl)ester;

2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazin. Examplesof water-soluble UVB filter substances include salts of2-phenylbenzimidazol-5-sulphonic acid including the sodium, potassium ortriethanolammonium salt and sulphonic acid; sulphonic acid derivativesof benzophenones, e.g., 2-hydroxy-4-methoxybenzophenon-5-sulphonic acidand its salts; sulphonic acid derivatives of 3-benzylidene camphor suchas 4-(2-oxo-3-bornylidene methyl)benzolsulphonic acid,2-methyl-5-(2-oxo-3-bornylidenemethyl)sulphonic acid and its salts.

UVA substances filter radiation in the range between 320 nm and about.400 nm, the co-called UVA range. Derivatives of dibenzoylmethane arepredominantly used to protect against rays in the UVA range and include,for example,1-(4′-tert.butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione and1-phenyl-3-(4′-iso-propylphenyl)propane-1,3-dione.

Sebaceous glands are components of the pilosebaceous unit. They arelocated throughout the body, especially on the face and upper trunk, andproduce sebum, a lipid-rich secretion that coats the hair and theepidermal surface. Sebaceous glands are involved in the pathogenesis ofseveral diseases, the most frequent one being acne vulgaris. Acne is amultifactorial disease characterized by the occlusion of follicles byplugs made out of abnormally shed keratinocytes of the infundibulum(upper portion of the hair follicle) in the setting of excess sebumproduction by hyperactive sebaceous glands. Various treatment modalitiesfor acne exist that aim in modifying the rate of sebum secretion by thesebaceous glands (e.g., retinoids), inhibiting the bacterial overgrowthin the follicular duct (antibiotics), or decreasing the inflammation ofacne lesions (anti-inflammatory agents). Most of these agents are notcurative of acne and simply control the disease by affecting one of theaforementioned pathogenic factors. Oral retinoids are a notableexception: they are potent drugs that can achieve a significant curerate for acne, but their side effect profile often limits their use.Advantages of the present invention include that treatment canpermanently alter the pilosebaceous unit, rendering it no longersusceptible to pore pluggage without the side effects associated withoral retinoids.

The term “sebaceous gland disorders” is intended to include thosesebaceous gland disorders which can be treated by a photosensitizedmaterial, such as PpIX that is converted from ALA. The PpIX can bephotodynamically activated, e.g., reactive, such that it is susceptibleto photoactivation or stimulation, e.g., light, i.e., laser stimulation.The activation or excitation of the material generates reactive species,such as a triplet state, which can interact with oxygen to producesinglet oxygen, causing membrane damage and cell destruction. Thesinglet oxygen can interact with the site of pore pluggage,inflammation, bacteria, viruses, etc. and promote, for example,oxidation of those agents which are associated with the disorder.Examples of sebaceous gland disorders which can be treated by themethods of the invention include sebaceous gland hyperplasia, acnevulgaris and acne rosacea. Of particular importance is treatment of acneby the method of the invention.

The term “pluggage” is intended to obstruction of the pores by thebuildup of sebum, dirt, bacteria, mites, oils, and/or cosmetics in thepore, e.g., about the infundibulum.

The term “acne” is art recognized and is intended to include acnevulgaris and acne rosacea. Acne vulgaris the most common skin diseaseseen in dermatologic practice which affects approximately 17 millionpeople in the United States. Its precise cause is unknown, althoughabnormal keratin production with obstruction of the follicular opening,increased production of sebum (lipids secreted by the androgen-sensitivesebaceous glands), proliferation of Propionibacterium acnes (anaerobicfollicular diphtheroids), follicular rupture and follicular mites(demodex) are commonly associated with acne.

Skin conditions such as acne are believed to be caused or exacerbated byexcessive sebum flow produced by sebaceous glands most of which areadjacent to and discharge sebum into, hair follicles. Sebum is composedof keratin, fat, wax and cellular debris. Sebum forms a moist, oily,acidic film that is mildly antibacterial and antifungal and may to someextent protect the skin against drying. It is known that the bacteriawhich contribute to acne, Propionibacterium acnes or (P-acnes), grows insebum. Significant sebum flow in humans begins at puberty. This is whenacne problems generally arise. The methods of the present inventiondecrease or eliminate the overproduction of sebum and thereby eliminatesacne.

Not to be limited by theory, photodynamic stimulation of aphotosensitizing agent, e.g., PpIX, can cause oxidation anddecomposition of the unwanted material(s), thereby degrading andremoving unwanted material from the pore. Additionally, this treatmentcan also cause the opening to the infundibulum to become modified, e.g.,the pore opening is enlarged. Consequently, alteration of the poreopening, such as enlargement of the pore opening, a change in the poreshape, or enlargement of the pore opening prevents unwanted dirt,bacteria, viruses and/or oils from building up in the treated area,e.g., the infundibulum.

Preferably, the energy source produces an exposure area of between about3 to about 100 millimeters to treat a section of skin afflicted with asebaceous gland disorder, as described above. The fluence is limitedsuch that the skin is not damaged while the sebaceous gland disorder istreated, e.g., eradicated, inhibited, or prevented. The fluence iscontrolled such that localized destruction to the undesired sebaceousgland disorder occurs with little or no non-specific necrosis ofsurrounding tissue.

Suitable energy sources include light-emitting diodes, incandescentlamps, xenon arc lamps, lasers or sunlight. Suitable examples ofcontinuous wave apparati include, for example, diodes. Suitable flashlamps include, for example pulse dye lasers and Alexandrite lasers.Representative lasers having wavelengths strongly absorbed by PpIX,within the epidermis and infundibulum, or sebaceous gland, include theshort-pulsed green dye laser (504 and 510 nm), yellow long-pulsed dyelaser (585-600 nm)the copper vapor laser (511 nm) and the Q-switchedneodymium (Nd):YAG laser having a frequency doubled wavelength using apotassium diphosphate crystal to produce visible green light having awavelength of 532 nm. Further examples of lasers which are suitable foruse as energy sources include those in the following table of lasers:

Types of Laser

Commercial Laser Types, Organized by Wavelength

Wavelength, mm Type Output type and power 0.532 Doubled Nd-YAG Pulsed to50 W or CW to watts, pulsed or CW for 50 W average power 0.578 Coppervapor Pulsed, tens of watts 400-700 nm Pulsed Dye 0.1 to 10 Joules 514.5nm Ar Ion up to tens of watts 530.9 nm Kr Ion approximately 5 watts600-900 nm GaAlAs tens of watts depending semiconductor on design diodearray

The depth of penetration of the energy, e.g., light, emitted from theenergy source, such as a laser, is dependent upon its wavelength.Wavelengths in the visible to near IR have the best penetration and aretherefore best for use to treat the sebaceous gland and infundibulumlocated within the dermis.

For example, ALA, adapted to accumulate selectively in the infundibulumand/or the sebaceous gland, is first applied to the region of afflictedskin to be treated. Following absorption of the ALA, the ALA undergoesconversion to PpIX via the porphyrin synthetic pathway, is exposed to anenergy source, e.g., a laser, capable of producing a wavelength readilyabsorbed by the converted ALA, e.g., PpIX, thereby selectivelyphotodynamically treating those regions of the dermis known to havetrapped oils, bacteria, dirt, etc. i.e., the pilosebaceous unit, whichincludes the pore opening, infundibulum and sebaceous gland. Because thePpIX is selectively concentrated within or about these undesireddeposits, the deposits are degraded by photodynamically reactive speciesgenerated from the activated material. There is minimal to nodestruction of normal adjacent epidermal and dermal structures.

Preferably, the treatment of the invention modifies the pore opening tothe infundibulum such that the geometry, e.g., the shape, of the openingis permanently altered. Adjustment of the concentration of the ALA andthe amount of energy applied by the energy source effects the increasedopening size of the pore, thereby preventing accumulation of dirt, oils,and/or bacteria, in that follicle. The operator will need to assess theparameters to illicit the desired effect and will be determined on apatient by patient basis. Generally, it is most desirable to alter theshape of the pore, leaving the pore enlarged and no longer prone tobuildup of sebum and/or foreign materials which would cause porepluggage.

As previously stated, the present invention involves the use of energysources, e.g., lasers, to target sebaceous glands and cause theirphotodynamic alteration, e.g., diminishment in size. Sebaceous glandsare mainly composed of amorphous lipid material and do not containeffective amounts of PpIX to cause photodynamic stimulation of thetissue/gland/pathogenic species. In order to achieve selectivephotodynamic treatment of sebaceous glands and confine the extent of anyinjury in the surrounding tissue, topically applied ALA with selectivedistribution to the pilosebaceous unit can be utilized. The introductionof ALA in sebaceous glands followed by exposure to energy (light) with awavelength that corresponds to the absorption peak of the PpIX, willincrease the local absorption of light in tissue and lead to selectivephotodynamic damage of sebaceous glands.

The infundibulum is a critical site in the pathogenesis of many of thedisease states, especially acne. There is evidence that abnormalproliferation and desquamation of infundibular keratinocytes leads tothe formation of microcomedones and, later on, to clinically visiblefollicular “plugs” or comedones. Clinically, it appears that somesebaceous follicles are more prone than others to develop acne lesions,possibly due to an inherent structural difference or functionalabnormality of the infundibulum, that predisposes them to form plugs andocclude. The self-resolving nature of acne in most patients may reflectthe elimination of such “acne-prone” follicles which are eventuallyreplaced by normal skin or fibrosis after repeated bouts ofinflammation. If the architecture of the infundibulum is important inthe pathogenesis of acne, then selective destruction of this portion ofthe follicle through ALA assisted energy, e.g., laser, targeting canhelp eliminate or correct the “pathologic” site by reshaping theinfundibulum so as to extrude any occluded material.

Delivery of ALA to the follicle matrix can be achieved by topicalapplication, injection, liposome encapsulation technology, massage,iontophoresis or ultrasonic technology, or other means for delivery ofcompounds into the dermal region of the skin, e.g., pharmaceuticallyacceptable carriers.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting ALA of the presentinvention within or to the subject such that it can performs itsintended function. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. Preferred carriers includethose which are capable of entering a pore by surface action and solventtransport such that the ALA is carried into or about the pore, e.g.,into the sebaceous gland, to the plug, into the infundibulum and/or intothe sebaceous gland and infundibulum.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening and perfuming agents, preservativesand antioxidants can also be present in the compositions.

Liquid dosage forms for topical administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, creams, lotions, ointments, suspensions and syrups. Inaddition to the active ingredient, the liquid dosage forms may containinert diluents commonly used in the art, such as, for example, water orother solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, peach,almond and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

The term “cream” is art recognized and is intended to include semi-solidemulsion systems which contain both an oil and water. Oil in watercreams are water miscible and are well absorbed into the skin, AqueousCream BP. Water in oil (oily) creams are immiscible with water and,therefore, more difficult to remove from the skin. These creams areemollients, lubricate and moisturize, e.g., Oily Cream BP. Both systemsrequire the addition of either a natural or a synthetic surfactant oremulsifier.

The term “ointment” is art recognized and is intended to include thosesystems which have oil or grease as their continuous phase. Ointmentsare semi-solid anhydrous substances and are occlusive, emollient andprotective. Ointments restrict transepidermal water loss and aretherefore hydrating and moisturizing. Ointments can be divided into twomain groups—fatty, e.g., White soft paraffin (petrolatum, Vaseline), andwater soluble, e.g., Macrogol (polyethylene glycol) Ointment BP.

The term “lotion” is art recognized and is intended to include thosesolutions typically used in dermatological applications.

The term “gel” is art recognized and is intended to include semi-solidpermutations gelled with high molecular weight polymers, e.g.,carboxypolymethylene (Carbomer BP) or methylcellulose, and can beregarded as semi-plastic aqueous lotions. They are typically non-greasy,water miscible, easy to apply and wash off, and are especially suitablefor treating hairy parts of the body.

In a one embodiment, liposomes are used to deliver ALA to the folliclematrix. Liposomes provide site-specific transdermal delivery to thefollicle matrix. In this embodiment, the ALA is microencapsulated withinthe liposome and topically applied to the epidermis of the skin.

As noted above, the carrier according to the present inventionpotentially involves encapsulating the effective amount of ALA within aspecific liposome to provide for efficient transdermal delivery of ALAthrough the layers of the skin. These liposomal compositions aretopically applied to the skin and deliver the encapsulated ALA to thefollicle region including the sebaceous gland and infundibulum.Following delivery of ALA, irradiation results in highly specifictargeting of the follicle matrix and destruction of oils, dirt,bacteria, mites, or viruses within the infected area.

Liposomes are microscopic spherical membrane-enclosed vesicles or sacks(0.5-500 mm in diameter) made artificially in the laboratory using avariety of methods. Within the scope of the present invention, theliposomes should be non-toxic to living cells and they should deliverthe contents, in this case ALA, into the follicle and immediatelysurrounding tissue. The liposomes according to the present invention maybe of various sizes and may comprise either one or several membranelayers separating the internal and external compartments.

The liposomes may be made from natural and synthetic phospholipids, andglycolipids and other lipids and lipid congeners; cholesterol,cholesterol derivatives and other cholesterol congeners; charged specieswhich impart a net charge to the membrane; reactive species which canreact after liposome formation to link additional molecules to thelysome membrane; and other lipid soluble compounds which have chemicalor biological activities.

A general discussion of the liposomes and liposome technology can befound in an article entitled, “Liposomes” by Marc J. Ostro, published inSCIENTIFIC AMERICAN, January 1987, Vol. 256, pp. 102-111 and in a threevolume work entitled, “Liposome Technology” edited by G. Gregorriadis,1984, published by CRC press, Boca Raton, Fla. the pertinent portions ofwhich are incorporated herein by reference.

Topically-applied ALA initially enters the infundibulum and later isdistributed to the sebaceous glands. It is possible to actively drivethe ALA into the follicles by massage, pressure, ultrasound, oriontophoresis, after topically applying the ALA to the skin surface. ALAcan be rapidly driven into sebaceous follicles and eccrine sweat ductsby iontophoresis. Wiping the surface with or without a solvent afterdelivery into the follicles, can be used to remove residual materialfrom the skin surface. Thus, after appropriate application and wiping,the ALA can be preferentially located in follicles, within theinfundibula or the infundibula and sebaceous glands.

For photodynamic effects, lower average irradiance exposures given overlonger exposure time would be appropriate for example approximately10-100 mW/cm2 delivered for about 100-2000 seconds (total fluence, 1-200J/cm2). For photodynamic effect, light sources such as light-emittingdiodes, incandescent lamps, xenon arc lamps, lasers or sunlight can beused.

In order to form and retain a plug within the infundibulum, there mustbe a constriction along the outflow tract. As material including sebum,cells, or bacteria accumulate and are concentrated onto the plug, wallsof the infundibulum are dilated until the middle or lower part of theinfundibulum is larger in diameter than its outlet (the surface pore).If the outlet diameter can be increased, the plug is more likely to beexpelled and pressure within the sebaceous follicle decreased beforerupture can occur. The upper region of the infundibulum is also thesource of follicular neck cells which shed into the infundibulum and addto the plug. For these reasons, the walls of the upper portion of theinfundibulum and especially its pore at the skin surface are the primarytarget for ALA-assisted sebaceous gland disorder treatment, e.g. acnetreatment. In a manner conceptually similar to laser skin “resurfacing”,the shape and size of the infundibulum and its outlet pore can beaffected by ALA-assisted photodynamic treatment. The dermis immediatelysurrounding sebaceous follicles, is largely responsible for maintainingshape of the infundibulum, and should be altered to produce a permanentaffect.

The invention is further illustrated by the following examples which inno way should be construed as being further limiting. The contents ofall references, pending patent applications and published patentapplications, cited throughout this application, including thosereferenced in the background section, are hereby incorporated byreference. It should be understood that the models used throughout theexamples are accepted models predictive of efficacy in humans.

EXAMPLES

Photodynamic therapy with topical ALA was tested for the treatment ofacne vulgaris, in an open-label prospective human study. Each of 22subjects with acne on the back was treated in 4 sites with ALA plus redlight (ALA-PDT), ALA alone, light alone, and untreated control. Half ofthe subjects were treated once; half were treated 4 times, 20% topicalALA was applied with 3 hr occlusion, and 150 J/cm² broad band light(550-700 mm) was given. Sebum excretion rate and auto-fluorescence fromfollicular bacteria were measured before, and at 2, 3, 10, and 20 weeksafter treatment. Histologic changes and PpIX synthesis in pilosebaceousunits were observed from skin biopsies. ALA-PDT caused a transientacne-like folliculitis. Sebum excretion was eliminated for severalweeks, and decreased for 20 weeks after PDT; multiple treatments causedgreater suppression of sebum. Bacterial porphyrin fluorescence was alsosuppressed by PDT. On histology, sebaceous glands showed acute damageand were smaller 20 weeks after PDT. There was clinical andstatistically significant clearance of inflammatory acne by ALA-PDT, forat least 20 weeks after multiple ALA-PDT treatments and 10 weeks after asingle treatment. Transient hyperpigmentation, superficial exfoliationand crusting were observed, which cleared without scarring. The presentmethods of the invention provide topical ALA-PDT as an effectivetreatment of acne vulgaris. ALA-PDT causes phototoxicity to sebaceousfollicles, prolonged suppression of sebaceous gland function, andapparent decrease in follicular bacteria after PDT.

Material and Methods Subject Selection

Twenty-two subjects of both sexes with mild to moderate acne vulgaris(grade 1-4) (Burke et al., 1984) on their backs were enrolled betweenOctober 1998 and March 1999. People were excluded if they had used anytopical acne treatment, systemic antibiotics in the past 2 weeks, orsystemic retinoids in the past 1 year. People were also excluded usingmedication that could exacerbate or alleviate acne, planning to haveexcessive sunlight exposure, with a history of keloid orphotosensitivity disorder, with Fitzpatrick's skin phototype V-VI, andpregnant or lactating women.

Study Design

Subjects were randomly divided into single treatment and multipletreatment groups. Each patient's back was equally divided into four7.5×10 cm areas for ALA plus red light (ALS-PDT), ALA alone, lightalone, and untreated control. Sites were marked withtemplates toprecisely relocate each test area. At baseline, clinical evaluations,natural bacterial porphyrin fluorescence photography, and sebumexcretion rate (SER) evaluation were performed. Before application ofALA, the skin was cleaned with 70% isopropyl alcohol. Then, 20% topicalALA in a hydroalcoholic vehicle (Levulan, a gift from DUSApharmaceutical) was applied for 3h under occlusion with plastic film(Saran wrap), and 150 J/cm² broad band light (550-700 nm) was given tothe ALA-PDT and light alone areas. In the multiple treatment group,subjects were treated once a week for 4 consecutive weeks. In thisgroup, if severe exfoliation, erosions or purpura occurred, treatmentwas postponed to the following week. In both groups, subjects returnedone week after treatments for clinical evaluations and at weeks 2, 3, 10and 20 for clinical, fluorescence, and SER evaluations.

Clinical Evaluations

Each subject's acne was visually assessed using an inflammatory acnescore modified from that previously described (Michaelsson et al.,1977). The modification used in this study accounted for both number andsize of acne lesions. The numbers of comedo, inflammatory comedo,papules, pustules, nodules and cysts in each test area were recorded.Each type of lesion was given a severity index as follows: 0.5 forcomedo (<1 mm), 0.75 for inflammatory comedo, 1 for papule (1 mm-5 mm),2 for pustule, 3 for nodule (>5 mm), and 4 for inflammatory cyst.

Clinical improvement was globally assessed by 3 dermatologists unawareof the status of treatment, who blindly graded changes in acne fromfixed-magnification clinical photographs, after being shown a small setstandardized series of training slides not used in the data evaluation.The grading scale was defined as −3 for >50% exacerbation, −2 for25⁺-50% exacerbation, −1 for 1⁺-25% exacerbation, 0 if unchanged, 1 for1⁺-25% improvement, 2 for 25⁺-50% improvement, 3 for 50⁺-75%improvement, 4 for 75⁺-99% improvement, and 5 for 100% improvement, ascompared to the baseline.

Fluorescence Photography

A Nikon E2N digital camera body with a Nikon 105 mm macro lens was used.A filter (Corion LL-5505) was placed on the lens to block light below550 nm. The excitation light source was composed of two synchronizedphotoflashes with Norman 400 watt-second lampheads (FT400/FT6), mountedon a stationary tower with angles of incidence of 60 degreesbilaterally. Two 400 nm bandpass filters with 5 nm bandwidth (CorionS40-400S) were placed on the flashes. By this method, the punctateorange-red fluorescence of hair follicles populated with P. acnes wasseen. Fluorescence emission has been attributed to bacterialcoproporphyrin III and protoporphyrin IX, and intensity of fluorescenceis related to the P. acnes population. Fluorescence photography wasperformed at week, 0, 2, 3, 10 and 20 in all sites. The number ofpunctate red flourescent dots was counted blindly for each test area.

Sebum Excretion Rate (SER) Measurement

Sebum-absorbent tape (Sebutapes, CuDerm Corp, Dallas, Tex.) is anon-invasive, easy and reproducible method to evaluate human sebumoutput. The subject's skins was shaved, then cleansed for 15 secondswith a cotton gauze soaked in 70% ethanol. When the skin was completelydry, a strip of Sebutape was adhered to each test site for an hour.After removal from the skin, the white tape was placed on a black cardfor image analysis. Small transparent spots due to sebum excretion fromfollicles were visualized as a black spot on the white background. A CCDcamera and digital frame grabber were used to capture images of thesebutape, then examined using a computer-assisted image analysis(IP-LAB) system. The percentage of sebutape area covered by sebum spots(black) was calculated. Sebutape assays of SER were done this way, atweeks, 0, 2, 3, 10, and 20 in all sites.

Adverse Effects

Adverse effects were scored by clinical evaluation of erythema, edema,loss of epidermis, hyperpigmentation, hemorrhage, vesiculation, andexfoliation on a visual-analogue scale from 0 to 3 (0=absent,2=moderate, 3=severe) for each finding. Subjective sensation of pain,burning, and itching was generally maximum about 10 minutes into lightexposure, and was ranked at that time and the end of treatment (1 hour),by subjects on a scale from 0 to 3 similar to above.

Histologic Examination

Punch biopsy specimens (4 mm) were taken immediately after PDT, a fewweeks after PDT, and at 20 weeks from both the untreated control andALA-PDT areas. Specimens were sectioned in either vertical or horizontalfashion and stained with hematoxylin and eosin, Fontana-Masson, andMasson-Trichrome stains. Histologic examination was performed.Cross-sectioned areas of sebaceous glands, representative sebocytes, andthe sebocyte nuclear area were measured from planimetric analysis ofserial sectioned specimens of the skin using a computer-assistedplanimetry system. Area of sebaceous gland and the cytoplasm/nucleararea ratio in sebocyte were calculated and compared between control andPDT areas at each follow-up. To determine the level of PpIX convertedfrom ALA in the pilosebaceous units, punch biopsy specimens were alsotaken from ALA-treated areas after 3 h occlusion as described above. Aseries of horizontal cross-sections of fresh-frozen specimens wasperformed, and localization of PpIX production was noted by fluorescencemicroscopy.

Histological examinations were performed to get a qualitative picture ofreaction to PDT. A total of 15 specimens were obtained. Eight biopsiesof PDT-treated areas were taken with accompanying specimens from thenon-treatment area: four from multiple PDT-treated areas at follow-up 5,one from a multiple PDT-treated area at follow-up 3, one from a singlePDT-treated area immediately after PDT, one from a single PDT-treatedarea at follow-up 3, and one from a single PDT-treated area at follow-up5. Seven biopsies were obtained without an accompanying specimen fromcontrol areas, and were analyzed for morphological changes due to PDT:two from single PDT-treated areas immediately after PDT, one from anacneiform lesion appearing at 3 days after PDT, one from a singlePDT-treated area at follow-up 2, one from a single PDT-treated area atfollow-up 5, and one from a multiple PDT-treated area at follow-up 3.

Statistical Analysis

Treatment effects were determined based on the following analyses: (1)comparing the scores from each follow-up visit to the baseline scoresusing paired t tests, (2) comparing the change from baseline among thefour treatment sites using paired t tests, (3) comparing the change frombaseline between the single treatment and multiple treatment groupsusing two-sample t-tests, and (4) comparing the change from baselinebetween the single treatment and multiple treatment groups using arepeated measures analysis to combine data from all follow-up visits.Statistical significance was defined as p value of less than 0.05.

Results

Of the 23 subject enrolled, 22 (17 males and 5 females) completed thestudy. One was dropped from the study because the patient's asthmanecessitated systemic steroid treatment, which is one of the exclusioncriteria. The age of patients completing the study ranged from 18 to 44years. Characteristics of the subjects in both groups are shown in Table1.

TABLE I SUBJECT CHARACTERISTICS IN BOTH GROUPS. Single treatmentMultiple treatment group group Age (year)   30 ± 8.74   27 ± 4.56 GenderM/F 9/2 8/3 Skin Type I 9.1% Type I 18.2% phototype Type II 27.3% TypeII 36.4% Type III 54.5% Type III 27.3% Type IV 9.1% Type IV 18.2%Disease history (year) 11.45 ± 8.38  11.27 ± 4.24  Previous systemicantibiotic 3 (27%) 4 (36%) treatment (number of subjects) Previoustopical antibiotic 3 (27%) 4 (36%) treatment (number of subjectsPrevious systemic isotretinoin 3 (27%) 2 (18%) treatment (number ofsubjects) Number of baseline comedo 4.39 ± 6.68 6.48 ± 8.24 Number ofbaseline  7.68 ± 11.24 4.27 ± 5.06 inflammatory comedo Number ofbaseline papules 6.23 ± 5.03 7.55 ± 5.67 Number of baseline papules 0.55± 0.90 0.43 ± 0.70 Number of baseline Nodules 0.48 ± 0.90 1.32 ± 2.36Number of baseline cysts 0.00 ± 0.00 0.00 ± 0.00

An impressive, acute eruption of inflammatory acneiform lesions wasobserved in the ALA-PDT sites only, in all patients (100%) in bothgroups, starting approximately 3-4 days post treatment (FIG. 1). Theinduced lesions were papules, pustules and nodules which lasted for 4days to 3 weeks in the single treatment group. In the multiple-treatmentgroup, subsequent treatments induced progressively less inflammatoryacne, such that almost no new acneiform lesions were observed aftertreatment 4.

Inflammatory Acne Score (FIGS. 2A, 3)

SINGLE TREATMENT GROUP: only the area treated with ALA-PDT showedimprovement in acne, which was statistically significant starting 3weeks after treatment. The other three areas (ALA alone, light alone,untreated) showed slightly worse acne not significantly different thanbaseline, for all visits. When comparing the change from baselinebetween the are treated with single ALA-PDT and the other three areas,the differences were statistically significant at 3, 10, 20 weeks.

MULTIPLE TREATMENT GROUP: There was obvious and statisticallysignificant improvement in acne at all follow-up visits after multipleALA-PDT treatment. There was no improvement in ALA-alone, light along oruntreated sites. Change from baseline was significantly greater at sitesof multiple ALA-PDT compared with the other three sites for all visits(p<0.05). At visit 2 only (week 2), there was a barely-significantimprovement in the area treated with ALA along compared to the untreatedarea (p=0.046).

COMPARISON BETWEEN SINGLE AND MULTIPLE TREATMENT GROUPS: MultipleALA-PDT treatments group showed significantly better improvement thanthe single ALA-PDT treatment group at the first 3 follow-up visits. Thisdifference diminished after week 3. No significant differences betweenmultiple and single treatment group were observed in the non-PDT siteswith respect to each individual visit. When data from all follow-upvisits were combined, the multiple ALA-PDT and multiple ALA alonetreatment sites had better improvement than the single treatment group(p<0.001 and P=0.007, respectively).

Global Clinical-Improvement Score (FIG. 2 b)

SINGLE TREATMENT GROUP: The ALA-PDT site showed significant globalimprovement starting week 3 and extending through week 20. The areawithout treatment, and the area treated with light alone, also showedimprovement reaching statistical significant at weeks 3 and 20 (p=0.017and 0.018), respectively. The difference between ALA-PDT and the otherthree treatment sites was statistically significant at weeks 3 and 10.

MULTIPLE TREATMENT GROUP: Significant improvement for the ALA-PDTrelated area was observed starting visit 1 (week 1) and this improvementpersisted throughout all of the 4 follow-up visits (up to 20 weeks atleast). The area treated with ALA alone at visit 2 and the area treatedwith light alone or ALA alone at visit 5 also showed improvementreaching statistical significance. However, there was a significantlybetter improvement in the ALA-PDT treated site than the other 3 sites,at all follow-up visits.

COMPARISON BETWEEN SINGLE AND MULTIPLE TREATMENT GROUPS: The multipleALA-PDT treatments group had significantly better improvement than thesingle ALA-PDT treatment group when evaluated at the first 2 follow-upvisits (week 1 & 2). The single-treatment group did not have significantbetter acne improvement than multiple treatment, at any time. When datafrom all follow-up visits were combined, the comparison between singleALA-PDT and multiple ALA-PDT reached statistical significance (p=0.008).

Fluorescence Photography Evaluation (FIGS. 2 c, 4)

SINGLE TREATMENT GROUP: Only the ALA-PDT-treated sites showedsignificant loss of fluorescence related to P. acnes, which lasted forall 4 follow-up visits. The differences between ALA-PDT and the other 3test sites were also statistically significant for all visits.

MULTIPLE TREATMENT GROUP: Again, only the ALA-PDT sites showedsignificant loss of P. acnes, fluorescence, starting at follow-up visit2. The sites treated with ALA alone or untreated had significantlygreater fluorescence than baseline, at week 10 and 20. The differencesbetween the ALA-PDT area and the other 3 test sites was statisticallysignificant for all visits.

COMPARISON BETWEEN SINGLE AND MULTIPLE TREATMENT GROUPS: The grouptreated four times with ALA-PDT had better, but not significant,improvement than those treated with single ALA-PDT. Combining data fromall visits, the difference in fluorescence related to P. acnes betweensingle and multiple treatment groups still did not reach statisticalsignificance (p=0.081).

Sebum Excretion Rate (SER) (FIGS. 2D, 5, 6)

SINGLE TREATMENT GROUP: Only the area treated with ALA-PDT showed asignificant decrease of SER, which was at weeks 2, 3, 20. The SER in thearea treated with PDT was also significantly lower than any other testsites, at each follow-up visit.

MULTIPLE TREATMENT GROUP: The ALA-PDT sites showed significant decreasein SER, at all follow-up visits. The differences between the areatreated with ALA-PDT and the other 3 areas were also statisticallysignificant at all 4 visits.

COMPARISON BETWEEN SINGLE AND MULTIPLE TREATMENT GROUPS: MultipleALA-PDT suppressed SER more than single ALA-PDT; however, the differencewas significant only at the longest follow-up time, 20 weeks. When datafrom all follow-up visits were combined, multiple ALA-PDT caused farlower SER than single ALA-PDT (p=0.001).

History

Marked focal histologic changes in pilosebaceous glands were observed inall samples treated by ALA-PDT. In the control (untreated) biopsyspecimens, all subjects had well-developed sebaceous gland with typicalround or oval locules. Immediately after ALA-PDT, there was a mixed,neutrophil-predominant infiltrate along pilosebaceous units andperivascular area, and retiform degeneration of sebocytes (FIG. 7).There was an apparent reduction of sebaceous gland size, with a meandecrease of 40% immediate after PDT. Epidermal changes were alsoobserved with epidermal necrosis, vacuolizaiton of keratinocytes fromthe mid stratum spinulosum to the stratum granulosum, and neutrophilicexocytosis. At 3 days after PDT, the acneiform lesions induced by PDTshowed large intraepiderman pustules, disruption of hair follicles, andfrank sebaceous gland destruction replaced by a mixed,neutrophil-predominant dermal infiltrate (FIG. 8). Reduction ofsebaceous gland size compared with untreated control area was observed 3weeks after both single and multiple ALA-PDT (30% vs. 55%). Focalvacuolization of sebocytes (FIG. 9), granulomatous reaction, andperifollicular fibrosis were also observed, although some of thesebaceous glands had regained a normal morphology with smaller sizerelative to control. Cytoplasm/nuclear cross-section area ratio insebocyte was reduced, relative to non-treatment, by 38% and 56% insingle and multiple PDT, respectively.

In the multiple treatment group, improvement contained longer into thefollow-up period, such that by the end of the study (20 weeks after thelast treatment), reduction of sebaceous gland size and sebocytecytoplasm/nuclear area were 45% (range from 15-80%) and 46% (range from39-80%) and 46% (range from 39-53%) respectively. At 20 weeks aftermultiple ALA-PDT treatments, there was complete destruction or markedatrophy of sebaceous gland lobules, with comparatively few sebocytespresent (FIG. 10 a). Frequently, a granulomatous reaction composed ofmultinucleated giant cells and histiocytic infiltrates was seen at theremnant of destroyed sebaceous glands (FIG. 10 b). Perifollicularfibrosis (FIG. 10 c), inflammation and spongiosis were seenoccasionally, but these findings were not constant. The epidermisappeared completely normal.

Twenty weeks after a single ALA-PDT treatment, there was only a slightreduction of sebaceous gland size (a mean decrease of 17%) without otherapparent morphological changes or infiltrates. The sebocytecytoplasm/nuclear area ratio was not reduced. Masson-trichrome stainedspecimens showed perifollicular fibrosis caused by single or multipleALA-PDT, and mild disarray of collagen bundles in the mid-reticulardermis by multiple ALA-PDT. Fontana-Masson stain showed higher epidermalpigmentation after ALA-PDT, and slightly more dermal melanophages(pigment incontinence) compared with untreated sites.

Fluorescence microscopy of fresh-frozen sections after ALA application,showed bright porphyrin fluorescence in epidermis and pilosebaceousunits, compared with untreated skin. There was brighter PpIXfluorescence in sebocytes than in the adjacent follicular epithelialcells (FIG. 11).

Adverse Effects

Erythema and edema were most intense about 10 minutes after thebeginning of PDT and subsided to lesser intensity by the end of thelight exposure. There was a substantial decrease in erythema and edemaby 1 hour after treatment, in both groups. Subjective reports of pain,burning, and itch were more severe at 10 minutes after starting PDT thanat the end of treatment. A burning sensation became more severe withsubsequent treatments and was the main complaint (9, 67%, 67%, and 73%of subjects at treatment 1, 2, 3, and 4 respectively). Itching was thenext frequent subjective side effect of subsequent treatments (73, 73,55, and 55% of subjects at treatment 1, 2, 3 and 4). In contrast tomultiple ALA-PDT, itching was the main discomfort in the single ALA-PDTgroup, and pain was the least.

Erythema, hyperpigmentation, and exfoliation were commonly seen afterPDT. Six patients in the multiple treatment group could not continue theweekly treatment scheme and had to postpone their next treatment: two attreatment 3 and 4, two at treatment 3, and 2 at treatment 4. Erythemaand hyperpigmentation faded away completely at 20 weeks in 82% and 91%of the single-treatment subjects, respectively. None of the subjects hadexfoliation after 3 weeks post treatment. One subject in the singleALA-PDT group developed blistering in the PDT site, after vigorousaerobic exercise while wearing a tight outfit the day after treatment.This area healed without scarring in 3 weeks. In fact, no site in anysubject had any scarring. Multiple PDT caused long-lastinghyperpigmentation with 55% of subjects still showing some degree ofpigmentation at 20 weeks after treatment. In 10% of multiple-treatmentsubjects, superficial but very prominent exfoliation was seen after 4treatments, with transient purpura (average 1 week) and partial loss ofepidermis.

Discussion

The present invention demonstrates by fluorescence photography thatALA-induced PpIX fluorescence is greater in acne lesions thansurrounding tissue (FIG. 12). P. acnes bacteria produce porphyrins tothe extent that red fluorescence is easily seen, and is correlated withP. acnes colonization of sebaceous follicles. Topical ALA-PDT can,therefore, have several modes of action for acne treatment. Directphotodynamic injury of sebaceous glands can inhibit sebum production;photodynamic killing of P. acnes cansterilize sebaceous follicles;follicular obstruction can be reduced by changing keratinocyte sheddingand hyperkeratosis. The present invention provides that topical ALA-PDThas potent effects on acne vulgaris. Blinded clinical assessment showedobvious and significant improvement of acne for at least 10 weeks aftera single ALA-PDT treatment, and for at least 20 weeks after 4treatments. Surprisingly, even nodular acne responded well and cysticacne induced by ALA-PDT resolved quickly and completely. ALA-PDT causedacute inflammation followed by partial or complete necrosis of sebaceousglands, producing a monomorphic acneiform eruption which appeared aftera few days and then subsided over several days to weeks. A similareruption often occurs after starting systemic retinoids, which likeALA-PDT, strongly inhibit sebaceous gland activity. Sebum excretion wasinhibited abruptly by ALA-PDT, then slowly and only partially recoveredby 20 weeks after 4 ALA-PDT treatments. Histologically, sebaceous glandswere smaller, and remained so long after ALA-PDT. Fluorescence of theendogenous porphyrins associated with P. acnes was also significantlydecreased after both single and multiple ALA-PDT, for at least 20 weeks.Not to be limited by theory, it is believed that antibiotic effects onP. acnes can be readily achieved by a single ALA-PDT treatment. Takentogether, the present invention provides that topical ALA-PDT inhibitsmultiple pathogenetic factors of acne.

The acneiform eruption which appears 3-4 days after the first ALA-PDTtreatment was a constant finding in this study. The mechanism for thiseruption is unknown. It is believed that ALA-PDT disrupts sebocyte andP. acnes membranes, activating complement and neutrophil migration intothe perifollicular area. Reactive oxygen species produced by neutrophilsplay a significant role in disrupting the follicular epithelium, whichis responsible for the inflammatory process of acne. In addition, P.acnes activates complement and produces C5a, a potent neutrophilchemotactic factor. The bacterial cell wall peptidoglycan-polysaccharidesubstance may also lay a role in stimulating an immune granuloma typereaction, which was seen in this study following multiple ALA-PDT. Inthe multiple-treatment group, each subsequent ALA-PDT treatment produceda progressively less inflammatory and weaker acneiform eruption, whichis at least consistent with sterilization of the follicles by the firsttreatment.

ALA-PDT is a simple procedure, and has very little side-effects.Fortunately, there was no scarring in this study, after a total of about60 PDT sessions in 23 subjects. However, each treatment takes time, cansometimes be painful or pruritic, can cause erythema and edema,occasionally causes blistering and purpura, causes an acute acneiformeruption, and usually leads to hyperpigmentation which fades graduallyover weeks to months. To most people, these side-effects would betolerable in practice when ALA-PDT were to provide a permanentimprovement in acne. This situation is not unlike the use of systemicretinoids, which produce both long-lasting benefits and majorside-effects.

Those of ordinary skill in the art will recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto specific embodiments of the invention described specifically herein.Such equivalents are intended to be encompassed in the scope of thefollowing claims.

1. A method for the treatment or prevention of a sebaceous glanddisorder, comprising the steps of: topically applying an effectiveamount of a composition having at least one of 5-aminolevulinic acid, asalt of 5-aminolevulinic acid, and a pharmacologically equivalent formof 5-aminolevulinic acid to a skin surface, wherein the composition isconverted into a photosensitizing agent that is able to be activated byenergy which penetrates outer layers of epidermis, thereby enabling asufficient amount of the composition to infiltrate a pilosebaceous unitdistal of the skin surface; and exposing the infiltrated section of skinto light in the range of about 1 J/cm² to about 100 J/cm² to cause thephotosensitizing agent to become photodynamically activated.
 2. Themethod of claim 1, wherein the sebaceous gland disorder is at least oneof acne vulgaris, acne rosacea, and sebaceous gland hyperplasia.
 3. Themethod of claim 1, wherein the light is in the range of about 1 J/cm² toabout 50 J/cm².
 4. The method of claim 1, wherein the light comprisessunlight.
 5. The method of claim 4, wherein the sunlight is in the rangeof about 1 J/cm² to about 50 J/cm².
 6. The method of claim 1, wherein aconcentration of the composition is in the range of about 0.1 percent byweight to about 10 percent by weight.
 7. The method of claim 1, furthercomprising the step of topically applying a substance which absorbs UVradiation in the UVA or UVB range to the skin surface.
 8. The method ofclaim 7, wherein the composition is combined with the substance whichabsorbs UV radiation in the UVA or UVB range.
 9. The method of claim 8,wherein a concentration of the substance which absorbs UV radiation inthe UVA or UVB range is in the range of about 0.1 percent by weight toabout 30 percent by weight.
 10. The method of claim 7, wherein UVBfilters of the substance absorb energy between about 290 nm and about320 nm.
 11. The method of claim 7, wherein UVB filters of the substancecomprise at least one of a 3-benzylidene camphor, 4-aminobenzoic acid,cinnamic acid, salicylic acid, benzophenone, 2-phenylbenzimidazloe, andderivatives thereof.
 12. The method of claim 7, wherein UVB filters ofthe substance are oil soluble.
 13. The method of claim 7, wherein UVBfilters of the substance are water soluble.
 14. The method of claim 7,wherein UVA filters of the substance absorb energy between about 320 nman about 400 nm.
 15. The method of claim 7, wherein UVA filters of thesubstance comprise dibenzoylmethane and derivatives thereof.
 16. Themethod of claim 1, wherein the pharmacologically equivalent form of5-aminolevulinic acid comprises at least one of a pharmacologicallyequivalent amide of 5-aminolevulinic acid and a pharmacologicallyequivalent ester of 5-aminolevulinic acid.